Modified nucleoside analogs are an important class of available antineoplastic and antiviral drugs. Currently, there are no therapeutic compounds in use that are based on dimers of these nucleoside analogs. While dimers of the naturally occurring D-deoxyribofuranosyl nucleosides are well known, dimers in which one or both nucleosides are of the unnatural L-configuration are much less known, and their use in therapy of neoplastic and viral diseases is unknown.
In the synthesis of DNA-related oligomers, types of nucleoside dimers are synthesized as part of the overall process. These dimers usually include bases from naturally occurring DNA or RNA sequences. There is much known in the art about nucleoside monophosphate dimers. Many of these compounds have been synthesized and are available commercially. However, these dimers are made from nucleosides containing a sugar moiety in D-configuration.
Reese, C. B., Tetrahedron 34 (1978) 3143 describes the synthesis of fully-protected dinucleoside monophosphates by means of the phosphotriester approach.
Littauer, U. Z., and Soreg, H. (1982) in The Enzymes, Vol XV, Academic Press, N.Y., p. 517 is a standard reference which describes the enzymatic synthesis of dinucleotides.
Heikkilo, J., Stridh, S., Oberg,, B. and Chattopodhyaya, J., Acta Chem. Scand. B 39 (1985) 657-669, provides an example of the methodology used in the synthesis of a variety of ApG nucleoside phosphate dimers. Included are references and methods for synthesis of 3'.fwdarw.15' phosphates and 2'.fwdarw.15' phosphates by solution phase chemistry.
Gait, M., "Oligonucleotide Synthesis", IRL Press, Ltd., Oxford, England, 1984, is a general reference and a useful overview for oligonucleotide synthesis. The methods are applicable to synthesis of dimers, both by solution phase and solid phase methods. Both phosphitetriester and phosphotriester methods of coupling nucleosides are described. The solid phase method is useful for synthesizing dimers.
Gulyawa, V. and Holy, A., Coll. Czec. Chem. Commun 44 613 (1979), describe the enzymatic synthesis of a series of dimers by reaction of 2',-3' cyclic phosphate donors with ribonucleoside acceptors. The reaction was catalyzed by non-specific RNases. The donors are phosphorylated in the 5'-position, yielding the following compounds: donor nucleoside-(3'.fwdarw.15') acceptor nucleoside. Dimers were made with acceptors, .beta.-L-cytidine, .beta.-L-adenosine, and 9(.alpha.-L-lyxofuranosyl) adenine. Also, a large number of dimers with D-nucleosides in the acceptor 5'-position were made.
Holy, A., Sorm, F., Collect. Czech. Chem. Commun., 34, 3383 (1969), describe an enzymatic synthesis of .beta.-D-guanylyl-(3'.fwdarw.5')-.beta.-L-adenosine and .beta.-D-guanylyl-(3'.fwdarw.5')-.beta.-L-cytidine.
Schirmeister, H. and Pfleiderer, W., Helv. Chim. Acta 77, 10 (1994), describe trimer synthesis and intermediate dimers, all from .beta.-D-nucleosides. They used the phosphoramidite method which gave good yields.
Thus, dimers with L-deoxyribofuranosyl moieties in any position are new, as are dimers with L-ribofuranosyl moieties bonded to the 3'-position of the phosphate internucleotide bond.
Modified nucleoside analogues represent an important class of compounds in the available arsenal of antineoplastic and antiviral drugs. The anticancer agents 5-fluorodeoxyuridine (floxuridine), cytarabine and deoxycoformycin and the antiviral drugs 3'-azidodeoxythymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddI), acyclovir, 5-iododeoxyuridine (idoxuridine) fludarabine phosphate and vidarabine (adenine arabinoside/ara A) are representative of this class of monomeric nucleoside-derived compounds which are used therapeutically.
More recently, "antisense" oligonucleotide analogues with modified bases and/or phosphodiester backbones have been actively pursued as antiviral and antitumor agents. While no clinically approved drug has yet emerged from this class of compounds, it remains a very active field of research. Recently, antipodal L-sugar-based nucleosides also have found application as potent antiviral agents because they can inhibit viral enzymes without affecting mammalian enzymes, resulting in agents that have selective antiviral activity without concomitant mammalian cytotoxicity.
Most naturally occurring nucleosides have the D-configuration in the sugar moiety. While the chemical properties of L-nucleosides are similar to those of their .beta.-D-enantiomers, they exhibit very different biological profiles in mammalian cells and do not interfere with the transport of normal D-nucleosides. For example, .beta.-L-uridine is not phosphorylated at the 5'-position by human prostate phosphotransferase, which readily phosphorylates the enantiomeric .beta.-D-uridine. Apparently, L-nucleosides are not substrates for normal human cell kinases, but they may be phosphorylated by viral and cancer cell enzymes, allowing their use for the design of selective antiviral and anticancer drugs.
Oligonucleotides based on L-nucleosides have been studied previously. Octamers derived from .alpha.- and .beta.-L-thymidine were found resistant to fungal nucleases and calf spleen phosphodiesterase, which readily degrades the corresponding .beta.-D-oligonucleotide. Fujimory, et al, S. Fujimory, K. Shudo, Y. Hashimoto, J. Am. Chem. Soc., 112, 7436, have shown that enantiomeric poly-.alpha.-DNA recognizes complementary RNA but not complementary DNA. This principle has been used in the design of nuclease-resistant antisense oligonucleotides for potential therapeutic applications.
Thus, L-nucleoside-based compounds have potential as drugs against neoplastic and viral diseases. While L-sugar-derived nucleosides and their oligonucleotides have been widely evaluated for such activities, little is known regarding the biological activities of shorter oligomers such as dimers obtained by L-nucleoside substitution.
This invention comprises novel L-nucleoside-derived therapeutic antitumor and antiviral agents. Novel L-nucleoside-derived dinucleoside monophosphates, based on L-.alpha.-5-fluoro-2'-deoxyuridine showed a remarkably high potency activity profile in in vitro anti-cancer assays, with indications of unique mechanisms of action, including inhibition of telomerase. Therefore, the L-nucleosides can serve as building blocks for new drugs with the special advantage of low toxicity.